Förster resonance energy transfer (FRET) microscopy is a technique that provides the spatial and temporal resolution necessary to investigate protein-protein interactions in live specimens. One of the most robust ways of quantifying FRET is to measure the fluorescence lifetime of the donor fluorophore. Fluorescence lifetime imaging microscopy (FLIM) is routinely used in the biological sciences to monitor dynamic signaling events inside living cells. However, the demands of studying complex signaling networks in living cells using FRET-based biosensor probes requires the ability to track more than one donor fluorophore at the same time. Here, we demonstrate a novel (Pulsed Interleave Excitation) PIE-FastFLIM technique that can be used to simultaneously measure two FRET-based biosensor probes at the same pixel. The PIE-FastFLIM configuration uses tunable wavelengths and synchronized gating detection, which can be tailored and optimized for each biosensor probe. When combined with the model-free phasor plots analysis, the PIEFastFLIM approach enables quantitative multiplexed FRET measurements for tracking multiple signaling events simultaneously in live cells.
The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.
The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with
exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy
transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring
dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps
changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic
protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined
with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving
transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The
heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin
through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α
as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor
CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in
mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic
interactions in pituitary specific gene regulation.
Orange fluorescent proteins (FPs) are attractive candidates as Förster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.
The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET)
microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Currently,
the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs.
However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the
characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has
advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we
characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET
standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and
acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the
fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting
from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and
lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red
FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this
observation.
Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein
interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated
a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However,
the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy.
Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one
donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three
chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three
FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually
dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the
mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP
to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single
living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different
FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on
the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor
photobleaching FRET microscopy.
The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells.
We have witnessed remarkable advances over the past decade in the application of optical techniques to visualize the genetically encoded fluorescent proteins (FPs) in living systems. The imaging of the FPs inside living cells has become an essential tool for studies of cell biology and physiology. FPs are now available that span the visible spectrum from deep blue to deep red, providing a wide choice of genetically encoded fluorescent markers. Furthermore, some FPs have been identified that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells. These additions to the FP toolbox are now being used for some very innovative live-cell imaging applications. Here, we will highlight the characteristics and uses of a few of these exceptional probes. Many different optical methods can be combined with the FPs from marine organisms to provide quantitative measurements in living systems.
The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.
Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPa), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPa was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPa was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.
Regulated gene transcription is dependent on the steady-state concentration of DNA-binding and coregulatory proteins assembled in distinct regions of the cell nucleus. For example, several different transcriptional coactivator proteins, such as the Glucocorticoid Receptor Interacting Protein (GRIP), localize to distinct spherical intranuclear bodies that vary from approximately 0.2-1 micron in diameter. We are using multi-spectral wide-field microscopy of cells expressing coregulatory proteins labeled with the fluorescent proteins (FP) to study the mechanisms that control the assembly and distribution of these structures in living cells. However, variability between cells in the population makes an unbiased and consistent approach to this image analysis absolutely critical. To address this challenge, we developed a protocol for rigorous quantification of subnuclear organization in cell populations. Cells transiently co-expressing a green FP (GFP)-GRIP and the monomeric red FP (mRFP) are selected for imaging based only on the signal in the red channel, eliminating bias due to knowledge of coregulator organization. The impartially selected images of the GFP-coregulatory protein are then analyzed using an automated algorithm to objectively identify and measure the intranuclear bodies. By integrating all these features, this combination of unbiased image acquisition and automated analysis facilitates the precise and consistent measurement of thousands of protein bodies from hundreds of individual living cells that represent the population.
Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy coupled with the development of new fluorescent probes provide the tools to study protein interactions in living specimens. Spectral bleed-through or cross talk is a problem in one- and two-photon microscopy to recognize whether one is observing the sensitized emission or the bleed-through signals. In contrast, FLIM (fluorescence lifetime imaging microscopy) or lifetime measurements are independent of excitation intensity or fluorophore concentration. The combination of FLIM and FRET will provide high spatial (nanometer) and temporal (nanoseconds) resolution when compared to steady state FRET imaging. Importantly, spectral bleed-through is not an issue in FLIM imaging because only the donor fluorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the FRET efficiency and the distance between donor- and acceptor-labeled proteins. Moreover, the FRET-FLIM technique allows monitoring more than one pair of protein interactions in a single living cell.
The physics and chemistry of fluorescent resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years, but only with recent technical advances has it become feasible to apply FRET in biomedical research. FRET microscopy is a better method for studying the structure and localization of proteins under physiological conditions than are X-ray diffraction, nuclear magnetic resonance, or electron microscopy. In this study, we used four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides 2-D spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleed through and photo bleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. We will discuss the advantage of fluorescence lifetime methods to measure FRET signals at the moment of the protein-protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution.
Cells respond to environmental cues or developmental programs by modifying protein complexes in the nucleus to alter patterns of gene transcription. Recent advances in digital imaging coupled with the development of new fluorescent probes provide the tools to begin to study where and when changes in protein interactions take place in the nucleus of the living cell. Here, we describe the application of fluorescence resonance energy transfer (FRET) using both wide-field and 2-photon (2P) microscopy to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. The efficiency of FRET will be improved if the overlap of the donor emission spectra with the absorption spectra for the acceptor is increased. The trade off for this improved efficiency, however, is that there will be an increase in the background signal from which the weak sensitized acceptor emission must be extracted. Here, we compare and contrast the FRET signals obtained from dimerized C/EBP(alpha) proteins fused to several different color variants of the jellyfish green fluorescent protein (GFP). We use both wide-field and 2P FRET microscopy to characterize the spectral cross-talk and FRET signals for each of the donor and acceptor pairs.
KEYWORDS: Fluorescence resonance energy transfer, Proteins, Green fluorescent protein, Luminescence, Image filtering, Image fusion, Optical filters, Imaging systems, Signal detection, Energy transfer
Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.
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